A nucleic acid ligand (aptamer) is a nucleic acid macromolecule (e.g., DNA or RNA) that binds tightly to a specific molecular target. Like all nucleic acids, a particular nucleic acid ligand may be described by a linear sequence of nucleotides (A, U, T, C and G), typically 15-40 nucleotides long. In solution, the chain of nucleotides forms intramolecular interactions that fold the molecule into a complex three-dimensional shape. The shape of the nucleic acid ligand allows it to bind tightly against the surface of its target molecule. In addition to exhibiting remarkable specificity, nucleic acid ligands generally bind their targets with very high affinity, e.g., the majority of anti-protein nucleic acid ligands have equilibrium dissociation constants in the picomolar to low nanomolar range.
Nucleic acid ligands are generally discovered using an in vitro selection process referred to as SELEX (Systematic Evolution of Ligands by EXponential enrichment). See for example Gold et al. (U.S. Pat. No. 5,270,163). SELEX is an iterative process used to identify a nucleic acid ligand to a chosen molecular target from a large pool of nucleic acids. The process relies on standard molecular biological techniques, using multiple rounds of selection, partitioning, and amplification of nucleic acid ligands to resolve the nucleic acid ligands with the highest affinity for a target molecule.
While successful at eventually generating high affinity nucleic acid ligands, the SELEX process requires multiple time consuming rounds of selection, partitioning, and amplification, because during nucleic acid ligand selection, low affinity nucleic acid ligands are at an increased concentration in a nucleic acid ligand library compared to high affinity nucleic acid ligands. SELEX requires multiple rounds to isolate the high affinity nucleic acid ligands because the low affinity nucleic acid ligands must be eliminated gradually to ensure eventual selection of the high affinity nucleic acid ligands.
There is an unmet need for methods that can more efficiently discover nucleic acid ligands to target molecules.